Opiates and opioid peptides act by binding to specific receptor binding sites which are coupled to second messenger systems in neuronal cell membranes. One such second messenger system is opiate-mediated inhibition of adenylate cyclase, in which receptors are coupled to adenylate cyclase through specific auanine nucleotide binding proteins. This activity can now be measured reproducibly in brain membranes by a technique of low pH pretreatment which selectively eliminates stimulation of adenylate cyclase by other receptors but increases inhibition of adenylate cyclase by opiate agonists. This technique also increases the regulation of opite agonist binding sites by guanine nucleotides. This project will study the molecular mechanisms in brain membranes that regulate the coupling of opiate receptor to adenylate cyclase. First, it will explore the pharmacological properties of opiate-inhibited adenylate cyclase and determine the nature of the opiate receptor subtypes involved in this second messenger system. Second, this research will study the biochemical factors which regulate the activity of this system: by labeling the guanine nucleotide binding proteiins in brain membranes, it will determine the nature of the low pH effect which alters the balance between stimulated and inhibited adenylate cyclase. Finally, the project will focus on the effects of opiate tolerance on this second messenger system. Opiate tolerane does not appear to involve a loss of opiate receptor binding sites; however, it may involve a loss of coupling between receptors and adenylate cyclase. These studies will examine this possibililty and search for possible biochemical changes in quanine nucleotide binding proteins that may occur in response to tolerane. This research will not only help describe some of the molecular mechanisms of tolerance in brain, but will also help to explain acute actions of opiates by exploring the factors in brain membranes that regulate expression of opiate-inhibited adenylate cyclase.